Enzyme Kinetics and Glycan Structural Characterization of Secreted Alkaline Phosphatase Prepared Using the Baculovirus Expression Vector System

Authors
Zhang, Fuming
Murhammer, David W.
Linhardt, Robert J.
ORCID
https://orcid.org/0000-0003-2219-5833
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Issue Date
2002-07-02
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Full Citation
Enzyme Kinetics and Glycan Structural Characterization of Secreted Alkaline Phosphatase Prepared Using the Baculovirus Expression Vector System, F. Zhang, D.W. Murhammer, R.J. Linhardt, Applied Biochemistry and Biotechnology, 101, 197-210, 2002.
Abstract
Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that Vmax and Km of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the Vmax and Km of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man(2-8)GlcNAc2 and FucMan(3 or 4)GlcNAc2) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man4GlcNAc2 and FucMan4GlcNAc2 were the major rSEAP N-glycans produced in Sf-9 cells, while Man2GlcNAc2 was the major rSEAP N-glycan produced in Tn-5B1-4 cells.
Description
Applied Biochemistry and Biotechnology, 101, 197-210
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Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
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The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology
https://harc.rpi.edu/
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